The goal of this tudy is to provide arationale, on a molecular basis, as to the reason why certain metabolically important flavoenzymes contain a covalent 8alpha-substituted flavin coenzyme while others contain an unsubstituted flavin coenzyme which is bound to the apoenzyme by non-covalent interactions. The approaches used in this study will be three-fold and will focus on the 8alpha-N(3)- and 8alpha-N(1)-histidylflavins and the enzymes that contain these functionalities. The first approach will be an in-depth study of the pH-dependence of oxidation-reduction potentials of both model and enzyme-bound 8alpha-histidylflavins to determine if 8alpha-histidyl ionization is involvedin modifying the electron affinity of the isoalloxaine ring either in 1-electron or 2-electron redox reactions. The second approach is to use ENDOR spectroscopy to monitor both H and N coupling to the flavin semiquinone with the intent of determining whether any spin density is distributed on the 8alpha-imidazole substituent in both model and enzyme systems. The third approach will be a detailed comparative mechansistic study of two flavoenzymes which catalyze the same reaction (cholesterol oxidase) but differ in that one contains an 8alpha-N(1)-histidyl-FAD while the other contains "normal" FAD with the intent of correlating any mechanistic and redox differences to the influence of the 8alpha-N(1)-histidyl substituent. Mechanistic studies will also be extended on thiamine oxidase, a flaboenzyme that catalyzes a 4-electron oxidation and contains 8alpha-N(1)-histidyl-FAD.